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X chromosome inactivation results in the transcriptional silencing of one of the
two X chromosomes present in mammalian female cells. XIST expression from the
inactive X (Xi), and subsequent coating of the Xi by the XIST RNA, marks the initial
major observable characteristic of X-inactivation. The Xi then begins to exhibit features
of heterochromatin such as gene silencing, late replication, histone modification,
hypermethylation, and the recruitment of core histone variants. However, the exact
mechanistic events between XIST expression and the final heterochromatic state of the Xi
remain unclear.
To better understand the relationship between XIST, heterochromatin, and
silenced gene expression, the first goal of this study was to develop and characterize a
model system suitable for examination of those features of heterochromatin which can be
induced by XIST independent of gene silencing. Previous work using rodent/human
hybrids (containing active human and mouse X chromosomes) that had been
demethylated to reactivate XIST/Xist expression showed that while human XIST RNA
fails to localize properly to the human Xi, the mouse Xist localizes normally. In both
cases no silencing of X-linked genes was observed. To verify this hybrid cell system as a
model for studying the effects of XIST/Xist independent of inactivation, I examined the
DNA methylation, H3 acetylation, and gene expression statuses of multiple human and
mouse X-linked genes in these hybrids. The results confirm both i) the reactivation of
XIST/Xist due to demethylation, and ii) the lack of gene silencing despite the presence of
XIST/Xist.